KR100734612B1 - Production method of mevinolin from the soybean fermented with monascus sp - Google Patents

Abstract

A method of producing mevinolin by inoculating Monascus seed culture into a bean and fermenting is provided, which produces safe mevinolin containing no citrinin. A bean is inoculated with Monascus seed culture and fermented to produce the titled mevinolin. The Monascus seed culture is at least one selected from Monascus anka, Monascus purpreus, Monascus pilosus, Monascus ruber and Monascus kaoliang. The Monascus seed culture is obtained by inoculating Monascus sp. cultured in potato dextrose agar into a nutrient medium and culturing. The nutrient medium contains 2 to 8(w/v,%) of rice flour, 0.5 to 2.0(w/v,%) of peptone, 1 to 5(w/v,%), glycine and 6 to 20(w/v,%) of glucose and has an initial pH of 6.0±0.5. The bean is selected from a whole bean, bean flour or a processed product thereof.

Description

Production method of Mevinolin from the Soybean Fermented with Monascus sp.}

1 shows the chemical structures of mebinolin and citrinin. (A: lactone, B: mebinolinic acid (β-hydroxy acid), C: P - quinone citrinin, D: O - quinone citrinin)

Figure 2 shows HPLC chromatograms for citrinin, mebinolinic acid, mebinolin ethanol extract. (1, citrinin; 2, mebinolinic acid; 3, mebinolin; 4, methyl ester of mebinolinic acid; top: reference, bottom: sample)

3 is Monascus pilosus The effect of seed culture pH on mebinoline production by soybean fermentation of IFO 480 is shown. (All results are n = 3, values represent%, wt% / dry weight of fermented soybeans)

4 is Monascus pilosus The effect of fermentation time on mebinoline production by soybean fermentation of IFO 480 is shown. (All results are n = 3, values represent%, wt% / dry weight of fermented soybeans)

The present invention relates to a method for producing mebinoline of fermented soybeans, and more specifically, to inoculate and ferment soybean culture with soybean (baektae) to produce mebinolin, an anti-cholesterol-synthetic component, without producing citrinin, a fungal toxin. It is about how to produce.

Red yeast rice (red koji) is produced by inoculating grains of the genus Monascus , a red filamentous fungus, in natural food colorants, processed products, and digestion promoting agents in various regions of East Asia, mainly in China. It has been used for a long time as a material for improving blood flow.

Recently, the second metabolite produced by Rhodibacteria bacteria, mebinolin (or lovastatin, monacolin K, mevacor, C ₂₄ H ₃₆ O ₅ ), is a cholesterol biosynthesis enzyme HMG-CoA (3 -hydroxy-methyl-3-glutaryl-coenzyme) strongly inhibited reductase, and various functionalities have been reported according to blood lipid concentrations and cholesterol synthesis (Wang IK et al., J. Agri. Food Chem. 48: 3183- 3189 2000; Endo A. et al., J. Antibiotechnol. 38: 420-422 1985; Manzoni M & Rollini M. App.Microbiol.Biotechnol. 58: 555-564 2002; Wei W. et al., J. Nutri Biochem. 14: 314-318 2003).

But in the Monasker In addition to mevinolin as a metabolite, the strain is a pigment component such as monascorubramin or monascin (Lin CF. J. Ferment. Technol. 51: 407-414 1973; Lin TF & Demain AL. 36 : 70-75 1991) and produce mycotoxins such as citrinin, known as kidney and hepatic toxin components (Blanc PJ, Loret MO, Goma G. Biotechnol. Lett. 17: 291-294 1995; Wang YZ , Ju XL, Zhou YG.Food Microbiol. 22: 145-148 2005) Safety issues may arise with widespread use of the genus. That is, the production of these secondary metabolites is via the same polyketide pathway initiated by acetyl-coA and malonyl-coA in cells. Biosynthesis (Hendrickson L. et al., Chem. Biol. 6: 429-439 1999; Shinizu T. et al., Appl. Environ. Microbiol. 71: 3453-3457 2005).

Recently, Wang et al. Measured the amount of citrinin in cultures of several Monascus, and as a result, Monastus was detected at the highest 480 mg / L and the lowest 65 mg / L in all the samples examined. The need for a safety assessment for all products with strains was raised (Wang YZ, Ju XL, Zhou YG. Food Microbiol. 22: 145-148 2005).

Citrinin is a fungal metabolite that was discovered in 1931 and has been known as a fungal toxin that affects the function of the kidneys and liver. In particular, it was separated from Penicillium citrinum and named as citrinin, but its presence was also present in metabolites of Aspergillus or Monascus fungi, 0.065-0.48 mg / Known as ml. Blanc et al. (Blanc PJ, Loret MO, Goma G. Biotechnol. Lett. 17: 291-294 1995) also included glucose, ethanol, acetate, etc. as the C source in the composition of a synthetic medium for culturing M. ruber . Citrinin was not produced when using, but the production of citrinin was confirmed according to the type of medium when using wild strain Monascus louver ( M. ruber ). This means that the production of citrinin by the Monascus strain depends on the type of strain and the culture conditions. Especially when the water activity of the substrate is 0.8 or less (Comerio R. et al., Int. J. Food Microbiol. 42: 219-223 1998) or lack of aeration (Sabater-Vilar M. et al., J. Mutat. Res 444: 7-16 1999) Reported that the production of citrinin was inhibited suggests that the growth conditions of the strain are the biggest influencers.

 On the other hand, the mebinoline compounds present in the Monascus culture are largely divided into lactone and hydroxy acid types according to molecular structure as shown in FIG. 1 (Friedrich J. et al., J. Chromatogr A. 704: 363-367 1995; Li YG et al., J. Pharma. Biomed.Anal. 35: 1101-1112 2004). The lactone form of the pro-drug form is an active substance that exhibits pharmacological efficacy in vivo as the ring is opened to the physiologically active form of β-hydroxy acid by hydrolysis. drug) (Manzoni M. et al., Biotechnol. Lett. 21: 253-257 1999). Lactone-type mevinolin maintains a very stable form in pure methanol solution, but is easily converted to the active form of hydroxy carboxylate (open lactone) in water-containing substrates. It is known to maintain equilibrium in between. Therefore, Monascus fermentation, which is inevitable in water content, has been reported to be mainly composed of mebinolin in β-hydroxy acid form (Friedrich J. et al., J. Chromatogr. A. 704: 363-367 1995; Li YG et al., J. Pharma. Biomed.Anal. 35: 1101-1112 2004).

The lactone form of monacoline K is an inert compound of the same molecular structure as that of the statin drug (Lovastatin), discovered by Merk in 1979 in Aspergillus terreus and released as a drug for hyperlipidemia. Pro-drug (Manzoni M. et al., Biotechnol. Lett. 21: 253-257 1999). Statin drugs are very potent substances that directly inhibit the biosynthesis of endogenous cholesterol, which accounts for 80% of total cholesterol.However, the activation process of opening the molecular ring by carboxyesterase can damage liver and kidney as well as muscle. Side effects such as diseases (myopathy) (Thomson PD et al., JAMA. 289: 1681-1690 2003) are classified as specialty drugs sold only with a doctor's prescription. In particular, the activity of the carboxyesterase is known to be a lot of individuals because the individual difference is severe, many people (Paoletti R, Corsini A, Bellosta S. Atherosclerosis. 3: 35-40 2002).

Until now, most of the culture of red yeast rice was made by submerged fermentation of liquid culture, and mainly, optimum fermentation conditions, strain detection, and production of functional products containing red yeast rice have been studied. Solid state fermentation has also been attempted as a means of obtaining some functional ingredients, including pigments, but the main grains used in the medium are mostly rice, barley and wheat, and soybeans are successfully fermented by S. aureus. It is known that grains that are not suitable for fermented red yeast rice are unprecedented.

The present invention has been proposed to solve the problems of the prior art as described above, an object of the present invention is to inoculate and ferment red pepper culture medium in soybean (baektae) to produce a safe mebinoline containing no fungal toxin component citrinin To provide a way.

In order to achieve the above object, the present invention includes a method of inoculating and fermenting red hongkong bacteria culture medium soybean (baektae) to produce mebinoline.

In addition, the soybean refers to white soybean, and includes a method for producing mebinoline, characterized in that at least one or more selected from soybean (whole bean), soy flour, or processed products thereof.

In addition, the red yeast bacteria M. anka ( M. anka ), Monascus perpureus ( M. purpureus ), Monascus Philos ( M. pilosus ), Monascus louver ( M. ruber ), Monascus Kaoliang , M. kaling It includes a method for producing mebinoline, characterized in that at least one selected from.

In addition, Monascus among the erythrocytes ( M. pilosus ) IFO 480 includes a method for producing mebinoline characterized by being a superior strain.

In addition, the hongguk species culture medium includes a method for producing mebinoline, characterized in that the culture by inoculating the honggukyun cultured in a PDA medium in a liquid nutrient medium.

In addition, the liquid nutrient medium is rice flour 2 ~ 8 (w / v,%), peptone 0.5 ~ 2.0 (w / v,%), glycine 1 ~ 5 (w / v,%), glucose 6 ~ 20 (w / v,%) method of producing mebinoline characterized in that the preparation ratio and the medium of the initial pH 6.0 ± 0.5.

Hereinafter, the present invention will be described in detail.

In the present invention, soybean soybean fermentation was performed to explore the possibility of producing anti-cholesterol synthesizing component mevinolin and mold toxin citrinin using soybeans, which have not been tried so far. First, about 20 species of Monascus The strains of the genus were selected as the best strains producing mebinoline, the most suitable and functional metabolite, suitable for fermentation of red soybeans. In the next step, the production of secondary metabolite of hongkong soybean fermentation was evaluated according to the composition of the nutrient culture medium, and at the same time, the safe fermentation conditions were not detected. Rice, which was previously used as a substrate for fermentation of red yeast rice, has a higher composition of starch sugar that microorganisms can utilize than soybeans, so that it is easy to ferment in solid phase. No successful fermentation conditions have been established. In order to overcome this disadvantage, the present invention is to find the appropriate fermentation conditions by analyzing the use of the seed culture medium and the concentration of the optimal nutritional composition. Therefore, the most unique technique of fermented red soybean can be evaluated by the selection of excellent strains, the use of seed culture solution and the composition and ratio of the composition solution. Soybeans, not rice, were chosen as samples containing not only isoflavones, which are analogues of the plant's female hormone hormone estrogen, but also many functional compounds such as saponins, the main components of ginseng, and the unique unsaturated fatty acids (linoleic acid). It is one of the food materials with the highest physiological and economic utility value because of its high potential of bioactive ingredients for disease prevention and treatment functions other than primary nutritional function.

The present invention includes a method of inoculating and fermenting red hongkong bacterium culture medium with soybean (baektae) to produce mebinoline.

Herein, the soybean refers to white milk, and may be selected and used in at least one of whole beans, soy flour, or processed products thereof.

In the embodiment of the present invention for the fermentation of hongguk soybean beans were purchased as a sample of white baektae harvested in 2004 in Pyeongchang, Gangwon-do. In addition, 20 identified strains of the genus Monascus were distributed from Korea Food Research Institute and used to select excellent strains.

Soybeans can be soaked for 12-16 hours, then drained and about 100g (36-38% water content) of the sample in a 500ml Erlenmeyer flask, sealed with a ground, and sterilized can be used as a soybean sample.

Each erythrocyte is incubated in PDA (potato dextrose agar) medium at 30 ± 2 ° C. for 3-4 days, then a certain amount of culture (1-2 cm ² ) is taken and 4-5 times of sterile water is mixed and homogenized. . Homogenized Hong Kong bacteria culture is 5-10% (v / w) of the sample weight, aseptically inoculated into a solid medium (bean sample) and incubated for 15 ± 2 days, it is preferable to use the Monascus strain for selection. . In addition, inoculated with the homogenized hongguk culture in a liquid medium containing rice flour, peptone, glycine, glucose, and incubated for 3-4 days in a stirred incubator at 30 ℃ can be used as a hongguk seed culture solution in the present fermentation. The liquid medium for cultivation of hongguk species culture medium is rice flour 2-8 (w / v,%), peptone 0.5-2.0 (w / v,%), glycine 1-5 (w / v,%), glucose 6 It is preferable to use a medium having an initial pH of 6.0 ± 0.5, containing ˜20% (w / v) and the remainder of distilled water. In addition, the seed culture broth can be inoculated into the soybean sample can be fermented for 60 ± 5 days in an incubator at 30 ± 2 ℃. At this time, it is preferable to invert all cultures once a day from the third day of culture.

In solid phase fermentation of soybean of Monascus strain in the present invention, it was examined how the production of citrinin with mebinoline, a bioactive secondary metabolite, varies depending on the strain. Citrinin is a fungal metabolite, a fungal toxin that affects the function of the kidneys and liver.

According to the present invention, to produce mebinoline, the red yeast bacterium is M. anka , M. purpureus , M. pilosus , M. ruber , and M. ruber . , M. kaoliang , M. kaling It is preferable to use one or more of them. In addition, in the selection of the superior strain of the present invention, with the normal fermentation of hongkong soybean, the strain of mebinoline, which is a useful metabolite, is 0.05% or more and the detection amount of citrinin, which is a toxic component, is lower than 50 µg / g, M. anka IFO 478 (0.052%), M. purpureus ATCC 489 (0.051%), M. pilosus IFO 480 (0.072%), and M. kaoliang ATCC 595 (0.064%) strains. In particular, the content of mebinoline in the M. pilosus IFO 480 strain was 0.072%, which is 1.4 to 5.5 times higher than the rest of the strains, producing the most useful metabolites but not producing toxic citrinin. Is the best strain for fermenting red beans. In addition, as shown in Figure 2, the form of mebinoline present in the fermented red soybean is not the inactive lactone form, it was found that the main component is a mebinolinic acid compound, which is an active hydroxycarboxylate. Therefore, Nascus phylosus ( M. pilosus ) selected as a superior strain in the present invention Red yeast fermented soybeans using IFO 480 can be evaluated as a safe fermentation containing no fungal toxin component citrinin.

Hereinafter, the content of the present invention will be described in more detail with reference to Examples. However, these examples are only presented to understand the contents of the present invention, but the scope of the present invention is not limited to these embodiments.

Example 1 Preparation of Seed Culture Solution

Test Materials and Reagents:

Baektae harvested in 2004 was purchased from Pyeongchang, Gangwon-do, and used as a sample for fermentation. All solvents such as ethanol to extract the mebinoline compound were used for HPLC (Fisher Co., USA). The standard mevinolin (lactone form) and citrinin were purchased from Sigma (St. Louis, Mo., USA) and used as acidic mevinolin (hereinafter referred to as mebinolinic acid). (Alkali hydrolysis of Friedrich J. et al., J. Chromatogr. A. 704: 363-367 1995) was made with some modifications. That is, 0.1M NaOH was added to the standard product, and warmed at 50 ° C. for 1 hour, calibrated to pH 7.7 using 1M HCl, and then filtered (0.22 μm, Millipore Co., Bedford, MA, USA). It was analyzed by HPLC (JASCO PU-987 pump, Tokyo, Japan). Separation of mebinolin and mebinolinic acid from the standard was confirmed by UV spectra (UV spectra, λ max) and retention time ( t R).

Strains and medium used:

20 strains of the identified Monascus genera were distributed from Korea Food Research Institute and used for selection of superior strains. First, all the strains were incubated at 30 ° C. for 4 days using a PDA medium (potato dextrose agar; Difco, MI, USA), and then the suspension of spores was homogenized to inoculate the sample or directly to the seed culture.

Preparation of Seed Culture:

Lin medium, which has been widely used as a nutrient medium for solid state fermentation (Lin CF. J. Ferment. Technol. 51: 407-414 1973; Hajjaj H. et al., Appl. Environ. Microbiol. 67: 2596-2602 Various nutritional medium solutions of 2001) were modified as shown in Table 1 to prepare four types. Each strain isolated from the primary culture was inoculated with platinum to each type of preparation, and then incubated in a stirred incubator at 30 ° C. for 4 days and then used for the main fermentation.

Table 1 Medium Composition for Fermenting Soybean in Monascus

¹⁾ nutrients＼ ²⁾ type A  B C  D      rice powder 3.0 3.4 3.4 -      soy powder - - - 3.4      glucose - 12.9 12.9 12.9      peptone -  1.1 1.1 1.1      glycine -  2.6  2.6  2.6      sodium nitrate  0.1 - 0.2 0.2      magnesium sulfate  0.1 -  0.1  0.1

¹⁾ Weight / Volume:%

²⁾ liquid medium type

Example 2 Selection of excellent strain

Extraction and Quantitation of Mebinolin, Mebinolinic Acid, and Citrinin:

To 0.1 g of red yeast fermented soybean powder, add 1.0 ml of methanol, 70% ethanol, and 0.1% H ₃ PO ₄ acetonitrile, respectively, at 50 ° C. using an ultrasonicator (sonic & materials Inc. USA). The cells were crushed for hours and then centrifuged at 12,000 × g for 10 minutes. The supernatant was filtered (0.45 μm), and the content of secondary metabolites was measured using HPLC. A Capcell Pak C18 UG120 column (250 × 4.6 mm, SHISEIDO CO., Tokyo, Japan) was used. The solvent of the mobile phase eluted acetonitrile and 0.1% H ₃ PO ₄ at a ratio of 65:35 (v / v) and a flow rate of 0.8 ml / min. The search for the three compounds was simultaneously measured at 238 nm, the median wavelength of the maximum wavelengths 224, 237, 247 and 260 of each compound. The contents of mebinolin, mebinolinic acid, and citrinin were quantified using a calibration curve prepared using the standard of each compound.

Chromaticity Measurement:

70% ethanol was added to the powder of red yeast fermented soybean, stirred at room temperature for 1 hour, and diluted to an appropriate concentration (OD = 2.0 or less) to measure the chromaticity of the sample (Lin CF. J. Ferment. Technol. 51: 407-). 414 1973). That is, the color of the fermented product was evaluated by filtering with a filter of 0.45 ㎛ and then measured the absorbance at 500 nm (Hewlett packard spectrophotometer, model 8753, Germany).

Selection of good strains:

Monascus genus 20 strains were cultured in PDA medium, and then homogenized by mixing 5 times sterile water in 1.5 × 1.5cm strain culture, and taking 5% (v / w) of the sample weight soybean sample as a solid medium. Aseptically inoculated at 100g (sterile for 30 minutes at 121 ℃) and fermented for 15 days. Pseudomonas whether and Reviewing the secondary production of the metabolites of a normal fermentation according to the type of coarse strains, Pseudomonas coarse pure buffer As shown in Table 2 mouse (M. purpureus) Pseudomonas coarse fur pure mouse (M including ATCC 162 purpureus ) ATCC 228, M. ruber IFO 203, M. ruber Strains such as IFO 317 and M. species ATCC 435 were excluded from the treatment because of the low likelihood of normal fermentation in soybean media.

On the other hand, the detection amount of citrinin, known as a component of kidney and hepatotoxicity, was evaluated as the chromaticity value (OD ₅₀₀ nm) of the cells was high, that is, as a lot of red pigment metabolites were produced. The amount of citrinein detected or the color of the cell cultures that were not detected had a chromaticity value (OD ₅₀₀ nm) of 0.98 or less, and most of the visible colors were yellow or grayish brown. These results were cultured in YES (yeast extract sucrose) medium in 23 commercially available Monascus strains, and Wang et al. Reported that production of citrinin was detected in all samples regardless of the pigment of the culture (Wang YZ). , Ju XL, Zhou YG.Food Microbiol. 22: 145-148 2005). However, the strain culture of this experiment was different from the PDA medium of this experiment, using MEA (powdered malt extract, 20 g; peptone, 1.0 g; glucose, 20 g; agar, 15 g; distilled water to 1 L). The use of YES medium to prepare liquid cultures was compared with a marked difference. Thus Monascus The composition and concentration of the culture used to cultivate the strain can be evaluated as an important factor that has the greatest influence on the type and production of metabolites.

Along with the normal fermentation of red yeast beans, strains containing mebinoline, a useful metabolite, of 0.05% or more and a low detectable amount of toxic citrinin of 50 μg / g or less, were identified as M. anka IFO 478 ( 0.052%), M. purpureus ATCC 489 (0.051%), M. pilosus IFO 480 (0.072%), and M. kaoliang ATCC 595 (0.064%) strains were selected. In particular, the content of mebinoline in the M. pilosus IFO 480 strain was 0.072%, which is 1.4 to 5.5 times higher than the rest of the strains, producing the most useful metabolites but not producing toxic citrinin. Has been identified as the best strain for fermenting red yeast beans. In addition, as shown in Figure 2, the form of mebinoline present in the fermented red soybean is not the inactive lactone form, it was found that the main component is a mebinolinic acid compound, which is an active hydroxycarboxylate. This result is consistent with the report of Li et al. (Li YG et al., J. Pharma. Biomed. Anal. 35: 1101-1112 2004), and the main component of the red yeast rice ferment was found to be the acidic form of mebinolin K. .

In the present invention, more than 91.8% of the functional material contained in the pure solid fermented Hongguk fermented product has a possibility of being evaluated as a safe and functional material having high bioefficiency, since the active hydrolyzed mebinolinic acid is the main ingredient. Very high However, the total yield of mebinoline was less than 0.7 mg / g based on the dry sample weight of the fermentation, which was compared to a level lower than 400 mg / L, which is usually the maximum yield obtained through liquid culture (Lai LST et al. , Enzyme Microb.Technol. 36: 737-748 2005). Currently, KFDA's standard notification standard for red yeast fermented products suggests that the content of mebinoline and citrinin should meet the criteria of more than 0.05% and less than 50 ppb, respectively, in order for red yeast fermented products to be used as health functional foods.

Therefore, Monascus pilosus was selected as a superior strain in order to search for the optimum production conditions of fermented soybean fermented beans afterwards. After spawning 480 seedlings, secondary culture was performed in liquid medium prepared in four types as shown in Table 1 to enhance growth and proliferation of each strain. 10% (v / w) of the liquid culture was inoculated again into the soybean, a solid sample, and the amount of metabolite was evaluated while fermenting for a period of time.

TABLE 2 Monascus Production of mebinoline and citrinin according to strains (soybean fermentation for 15 days at 30 ℃)

Strain OD ₅₀₀ Mebinoline (%) ¹⁾ Citrinin (μg / g) M. anka IFO 478 1.55 0.052 21.5 M. anka IFO 873 0.98 0.019 ND M. pilosus IFO 520 0.12 0.015 ND M. pilosus IFO 480 0.23 0.072 ND M. purpureus ATCC 162 - ^-2) - M. purpureus ATCC 489 1.61 0.051 12.1 M. purpureus ATCC 228 - - - M. purpurea IFO 482 0.32 0.045 ND M. ruber IFO 203 - - - M. ruber IFO 492 0.20 0.031 ND M. ruber IFO 317 - - - M. araneosus IFO 483 1.45 0.032 19.3 M. kaoliang ATCC 592 0.51 0.017 ND M. kaoliang ATCC 595 1.27 0.064 31.2 M. kaling ATCC 598 0.37 0.013 ND M. sp. ATCC 435 - - - M. sp . ATCC 437 0.95 0.041 15.8 M. sp. IFO 301 0.31 0.019 ND M. sp. IFO 280 0.25 0.016 ND M. vitreus IFO 532 0.78 0.027 17.6

ND; Not detected

¹⁾ Mebinolin (%) = Mebinolin (%) + Mebinolinic Acid (%)

²⁾ fermentation failure

The detection limits of mebinolin and citrinin are 100 ng and 550 ng per gram, respectively.

All results were n = 3 and analysis was carried out according to the experimental method described.

Example 3 Influence of Seed Culture Composition

Using the soybean sample as a substrate, the seed culture solution, which is a liquid medium of four types of nutrient composition, was inoculated, and then fermented at 30 ° C. for 15 days. The results of measuring the amount of secondary metabolite produced by fermentation are shown in Table 3. That is, type B showed a total mebinoline content of 0.145%, and the yield was about two times higher than before the liquid nutrient medium was used. The production of citrinin was detected 71.5 μg / g in form D using soy flour instead of rice flour as the nutrient source of the liquid medium, but not in the remaining liquid broth fermentation. In particular, the total production of mebinoline with type C was 0.145% and 0.143%, respectively. In both types, citrinin was not detected, making it the most suitable liquid culture for solid phase fermentation of soybean. Was evaluated. In particular, the fermented soybeans inoculated with cultures of type A and D showed a small amount of secondary metabolites, suggesting that the fermentation of soybeans by the Monascus strain is an important condition for the nutrient source of the liquid culture.

Table 3 Monascus pilosus Secondary Metabolites of Soybean Fermentation by IFO 480

Type Mevinolin (%) Mevinolinic acid (%) Citrinin (㎍ / g) A - 0.017 ± 0.008 ND B 0.011 ± 0.003 0.134 ± 0.005 ND C 0.017 ± 0.004 0.126 ± 0.001 ND D - 0.101 ± 0.006 71.5

All results were n = 3 and analysis was carried out according to the experimental method described.

Lopez et al. (Lopez JLC et al., Enzyme Microb. It was. That is, the optimum ratio of C: N when using lactose as source C and soy meal or yeast extract as source N was reported to be 40 mg / g. In addition, Hajjaj et al. (Hajjaj H. et al., Appl. Environ. Changes emphasized the importance of strain culture conditions.

As shown in Table 1, the liquid culture of type B, except for sodium nitrate and magnesium sulfate in the composition of type C, rice powder (glucose), peptone (peptone) ), Glycine and the like were the same. Therefore, even without supplementing the trace minerals contained in the type C, the production amount of the functional metabolite of red yeast fermented soybean was found to be very good, and the liquid culture used for the solid phase fermentation thereafter was prepared and tested in the form of B. The composition of Form B is based on the amount of the preparation, rice powder 3.4 (w / v,%), peptone 1.1 (w / v,%), glycine 2.6 (w / v,%), glucose 12.9 ( w / v,%), and was incubated in a shaking incubator at 30 ° C. for 4 days, and then used as a seed culture solution to obtain red yeast fermented soybeans.

Example 4 Solid Phase Fermentation:

After soaking the soybean (white) for 12 hours, 100 g of the drained sample (water content 36-38%) was put in a 500 ml Erlenmeyer flask, sealed with a ground, and sterilized at 121 ° C. for 30 minutes. After homogenizing by mixing 5 times sterile water to a predetermined amount (1.5 × 1.5 cm) of the strain cultured in PDA medium, 5% (v / w) of the sample weight was taken aseptically inoculated into a solid medium. In addition, 10% (v / w) of the sample weight of each type of culture medium of Example 3 incubated with liquid medium was aseptically inoculated into the solid medium of the soybean sample. The inoculated sample was fermented for 60 days in an incubator at 30 ° C., followed by incubation for 10, 20, 30, 40, 50, and 60 days for each period, and then collected and observed the change of secondary metabolites of erythrocytes. At this time, all cultures were inverted once a day from the 3rd day of culture, and the collected samples were lyophilized and stored in a freezer until powdered use.

Example 5 Influence of Seed Culture pH and Fermentation Time

Effect of seed culture pH:

M. pilosus cultured first in PDA medium IFO 480 Platinum was inoculated into the liquid B-type culture, incubated for 4 days using a stirred incubator at 30 ° C, and then inoculated with 10% (v / w) of soybean (white) weight for 15 days at 30 ° C. Fermented. At this time, the initial pH of the liquid medium was adjusted to 4, 6, 8 using 0.1 N HCl and NaOH, and then sterilized for 15 minutes at a temperature of 121 ℃. The content of secondary metabolite of red yeast fermented soybean according to the pH of the liquid culture was as shown in FIG. 3. The amount of mebinoline contained in the fermentation of the sample inoculated with the culture at pH 6 was 0.213%, which was the highest production compared to 0.190% at pH 4 and 0.155% at pH 8 ( p <0.05). Was not detected.

Usually, the pH of strain culture is known to have an important effect on cell growth of filamentous fungi as well as on the production of secondary metabolites. That is, the activation conditions of specific enzymes involved in the production of mebinoline may be significantly affected by the acidity of the substrate (Lai LST et al., Enzyme Microb. Technol. 36: 737-748 2005; Sanchez S & Demain AL. Enzyme Microb.Technol. 31: 895-906 2002). Therefore, the subsequent culture medium was used to adjust the pH to 6.

Effect of fermentation time:

Liquid culture in solid medium of soybean samples (Type B, pH 6, M. pilosus IFO 480) was inoculated at 10% (v / w) of the sample weight, and then fermented for 60 days in an incubator at 30 ° C. as shown in FIG. 4. Overall, as the fermentation time passed, the production of mebinolin increased. Mebinoline production was measured at 0.12% on the 28th day of fermentation and increased to 0.22% on the 50th day of fermentation and then slowly decreased, but production did not stop until the end of fermentation. In general, biosynthesis of mebinoline is achieved by the use strain (Bang IY et al., Korean J. Food Sci. Technol. 35: 442-446 2003) and growth and reproduction conditions (Kwak EJ, Cha SK, Lim SI. Korean J. Food). Sci. Technol. 35: 830-834 2003; Hajjaj H. et al., Appl. Environ. Microbiol. 67: 2596-2602 2001). That is, the maximum amount of the metabolite was also observed when the maximum growth of the strain against the substrate is exhibited. As expected, the main components of mebinoline contained in fermented soybeans were acid-type mebinolinic acid, which accounted for 91.8-96.8%, and lactone-type mebinoline was only 3.2-8.2%. These results are indicative of mebinoline compounds obtained from fermented rice in red yeast rice (Friedrich J. et al., J. Chromatogr. A. 704: 363-367 1995; Li YG et al., J. Pharma. Biomed. Anal. 35 : 1101-1112 2004) suggests that acid-based mebinoline compounds should be used as reference materials in the future screening of functional materials of solid fermentation using erythrocytes.

Mebinolin and mebinolinic acids, and their esters of methyl, ethyl, propyl, etc., have generally been reported to be most well separated from the buffers of alcohols (Friedrich J. et al., J. Chromatogr. A. 704). : 363-367 1995). The mebinolin compound detected in the soybean fermentation product of the present invention was found to have a more stable form than the lactone type in the solvent condition of water-alcohol, and as shown in FIG. 2, the mebinolin compound of the mebinolinic acid and lactone type Only was found. Thus, Monascus pilosus IFO In hongguk fermented soybean using 480, an inhibitory component of endogenous cholesterol synthase (HMG-CoA), that is, a pharmacologically active acid type mebinolin (monacolin K, lovastatin) was identified as a main component.

Example 6 Citrineine Content of Red Yeast Fermented Soybean

In the present invention, in the solid phase fermentation of soybeans of the genus Monascus, it was examined how the production of citrinin varies with strains together with mebinoline, a bioactive secondary metabolite.

20 kinds of Monascus as shown in Table 2 Among the strains are M. anka , M. purpureus , Monascus araneosus , A portion of the angular hibiscus squall Liang (M. koliang) and Pseudomonas coarse bit Reus (M. vitreus) The production of citrinin was not confirmed in most strains except. The production of citrine was found to be very small, about 12.1-31.2 μg / g. In particular, the content of mebinoline in Monascus pilosus IFO 480 strain was 0.072%, which is 1.4 to 5.5 times higher than the rest of the strains, producing the most useful metabolites but not producing toxic citrinin. It was identified as the best strain for fermenting red soybeans. Therefore, Monascus pilosus selected as a good strain in this experiment Red yeast fermented soybean using IFO 480 was evaluated as a safe fermented product containing no fungal toxin component citrinin.

As described above, the present invention inoculates and ferments Hongkuk culture to soybean (baektae), which has not been attempted to ferment Hongkuk so far, to enable the production of an anti-cholesterol synthetic component mevinolin.

Claims (6)

M. anka , M. purpureus , M. pilosus , M. ruber , Monascus Kaoliang , M. kaling Method of producing mebinoline by inoculating and fermenting at least one selected honggyun species culture medium. The method of claim 1, wherein the soybean is at least one selected from soybeans, soy flour, or processed products thereof. delete The method of claim 1, wherein the red yeast fungus is M. pilosus A method of producing mebinoline characterized by an IFO 480. [Claim 2] The method for producing mebinoline according to claim 1, wherein the hongguk species culture medium is cultured by inoculating the honggukyun cultured in potato agar medium (PDA medium) in a liquid nutrient medium. The composition of claim 5, wherein the composition of the liquid nutrient medium is rice flour 2-8 (w / v,%), peptone 0.5-2.0 (w / v,%), glycine 1-5 (w / v,%), glucose 6 Method for producing mebinoline, characterized in that the preparation ratio of ˜20 (w / v,%) and the medium of the initial pH 6.0 ± 0.5.

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